3 replies to this topic

[scoob00]

Hi all,

This isn't homework, but I just cant seem to do it!

I am making a serial dilution, in the wells of a 96 plate. I need to be left with 75ul in each well after dilution. I would like a ten fold dilution across each well;
(2250ng protein/well, 225ng, 25ng, 2.5ng, 0.25ng)

So i had started off with 82.5ul of a 30ng/ul solution in my first well (giving 2475ng), and 75ul of dilutent in the rest of them. I have then transfered 7.5ul (225ng) to the second tube, and then 7.5ul to the next...

But this doesnt work - I only get the right concentration in the first well (2250ng), because of course I am only transfering to the second well the exact amount needed for that well (225ng) - not enough for the concentrations in the rest of the wells.

How do you work it out when a final volume is involved -eg my 75ul?? I am having a very stupid day...
Thanks...

Edited by scoob00, 19 May 2009 - 03:48 AM.

[DRT]

Thinking about it from this angle might help

Add 75ul diluent to each well except the first.
If the dilution is factor is 1/10 the diluent represents 9/10 and the final volume will be 75 / 0.9 = 83.333.  This is how much of the stock solution should be in the first well.
Transfer 83.3 - 75 = 83.3 / 10 = 8.33 to the second well ………..

[scoob00]

DRT, on May 20 2009, 01:05 AM, said:

Thinking about it from this angle might help

Add 75ul diluent to each well except the first.
If the dilution is factor is 1/10 the diluent represents 9/10 and the final volume will be 75 / 0.9 = 83.333.  This is how much of the stock solution should be in the first well.
Transfer 83.3 - 75 = 83.3 / 10 = 8.33 to the second well ………..

Hello DRT,

Thanks so much. That is by far the the easiest way I have ever seen it expained - I don't know why I struggle so much with this stuff, probably not a good sign for a phd student!!

so for 80ul final volume, it's simply; 80/0.9 = 88.8, - 80 = 8.88 to transfer.

or, if I want a 1/5 serial dilution;
diluent = 4/5 = 0.8
75 / 0.8 = 93.75 in first well.
93.75 - 75 = 93.75 / 5 = 18.75 to transfer.

and 1/3;
diluent = 2/3 = 0.666
75 / 0.666 = 112.6 in first well
112.6 - 75 = 112.6 / 3 = 37.5ul to transfer.


Thank you very much again,
Sccob  

Edited by scoob00, 20 May 2009 - 07:58 AM.

[medchemgirl]

This was very helpful for me too! It takes me a while calculating and confirming everything will be how I wanted. Now it just so easy!!!

scoob00, on May 20 2009, 03:42 AM, said:

DRT, on May 20 2009, 01:05 AM, said:

Thinking about it from this angle might help

Add 75ul diluent to each well except the first.
If the dilution is factor is 1/10 the diluent represents 9/10 and the final volume will be 75 / 0.9 = 83.333.  This is how much of the stock solution should be in the first well.
Transfer 83.3 - 75 = 83.3 / 10 = 8.33 to the second well ………..

Hello DRT,

Thanks so much. That is by far the the easiest way I have ever seen it expained - I don't know why I struggle so much with this stuff, probably not a good sign for a phd student!!

so for 80ul final volume, it's simply; 80/0.9 = 88.8, - 80 = 8.88 to transfer.

or, if I want a 1/5 serial dilution;
diluent = 4/5 = 0.8
75 / 0.8 = 93.75 in first well.
93.75 - 75 = 93.75 / 5 = 18.75 to transfer.

and 1/3;
diluent = 2/3 = 0.666
75 / 0.666 = 112.6 in first well
112.6 - 75 = 112.6 / 3 = 37.5ul to transfer.


Thank you very much again,
Sccob